In contrast to animals, plants continue to produce new organs throughout their life cycle. The above-ground organs are derived from the shoot apical meristem (SAM), which includes a pool of stem cells residing at the growing tip of the plant. Proliferating SAM cells produce an excess of daughter cells that are either incorporated into the developing leaf primordia at the SAM periphery or become part of the shoot. The core machinery controlling the progression of the cell cycle in plants, as well as in other eukaryotes, relies on the activity of cyclin-dependent kinases (Inze and De Veylder, 2006). Many aspects of cell cycle regulation are highly conserved among eukaryotes. It is, however, the integration of the basic cell cycle mechanisms with the developmental program that generates the enormous phenotypic variation among multicellular organisms, a process that is much less understood (Inze and De Veylder, 2006).
In contrast to the indeterminate SAM in Arabidopsis thaliana, leaves are determinate organs that have a defined morphology. Leaf development involves the concerted action of various hormone signalling pathways and transcription factor networks. Some of the major transcriptional regulators involved in the control of cell proliferation in leaves include AINTEGUMENTA (Mizukami and Fischer, 2000), PEAPOD (White, 2006), JAGGED (Dinneny et al., 2004; Ohno et al., 2004), BLADE ON PETIOLE (Ha et al., 2003), TCPs (Nath et al., 2003) and GROWTH-REGULATING FACTORs (GRFs) (Kim et al., 2003).
To obtain their characteristic final size and shape, growth of the developing leaf needs to be tightly coordinated first through cell proliferation and then by cell expansion (Piazza et al., 2005; Tsukaya, 2006). Initially, cell proliferation is observed throughout the developing leaf (Donnelly et al., 1999). Then, the cell cycle stops at the tip of the leaf and a mitotic arrest front moves towards the base of the organ (Donnelly et al., 1999). Once cells cease to divide, they begin to enlarge and cell growth becomes the driving force regulating organ size (Piazza et al., 2005; Tsukaya, 2006).
Currently, little is known about the molecular mechanisms that coordinate cell proliferation throughout a developing leaf. A known regulator is the TCP gene CINCINNATA (CIN), which controls the progression of the mitotic arrest front in snapdragon (Nath et al., 2003). Mutations such as cin (Nath et al., 2003) and triple knock-outs of its Arabidopsis homologues tcp2/4/10 (Schommer et al., 2008) cause changes in leaf morphogenesis and uneven organ curvature due to excess cell proliferation at the leaf margins. Interestingly, five Arabidopsis TCPs (TCP2, 3, 4, 10 and 24), as well as CIN, have a target site for microRNA (miRNA) miR319 (Palatnik at al., 2003). Overexpression of miR319 causes the degradation of these TCPs and the generation of crinkled leaves similar to those observed in tcp loss-of-function mutants (Palatnik et al., 2003). Mutations in the target site of the TCPs that diminish the interaction with the miRNA affect leaf morphology in Arabidopsis (Palatnik et al., 2003; Palatnik at al., 2007) and leaf complexity in tomato (Ori at al., 2007), and are lethal in extreme cases (Palatnik at al., 2003).
The GRF family of transcription factors comprises nine members in Arabidopsis (Kim et al., 2003). Seven of them have a target site for miR396 (Jones-Rhoades and Bartel, 2004). Loss-of-function mutations in different GRFs or overexpression of miR396, which decreases GRF levels, have been shown to reduce cell number in Arabidopsis leaves (Horiguchi et al., 2005; Kim et al., 2003; Kim and Kende, 2004; Liu at al., 2009). The GRFs work together with GRF-INTERACTING FACTORs (G/Fs), a small gene family encoding proteins with homology to the human SYT transcriptional co-activator (Horiguchi at al., 2005; Kim and Kende, 2004). Inactivation of GIF1 (Kim and Kende, 2004), also known as ANGUSTIFOLIA 3 (AN3) (Horiguchi at al., 2005), produces narrower leaves as a result of a reduction in cell proliferation.
It has been disclosed by Rodriguez at al., Development 137, 103-112 (2010), that a microRNA, miR396, plays a role in the coordination of cell proliferation in Arabidopsis leaves. They showed that in leaf primordia, miR396 is expressed at low levels, but its expression steadily increases during organ development. They showed that miR396 antagonizes the expression pattern of its targets, the GROWTH-REGULATING FACTOR (GRF) transcription factors. miR396 was shown to accumulate preferentially in the distal part of young developing leaves, restricting the expression of GRF2 to the proximal part of the organ. This, in turn, was shown to coincide with the activity of the cell proliferation marker CYCLINB1;1. miR396 was shown to attenuate cell proliferation in developing leaves through the repression of GRF activity and a decrease in the expression of cell cycle genes. Furthermore, they reported that over-expression of miR396 in a mutant lacking GRF-INTERACTING FACTOR 1 (GIF1) severely compromised the shoot meristem. miR396 was found to be expressed at low levels throughout the meristem, overlapping with the expression of its target, GRF2. In addition, it was shown that overexpression of miR396 can reduce cell proliferation and the size of the meristem. Arabidopsis plants with an increased activity of the transcription factor TCP4, which reduces cell proliferation in leaves, were shown to have higher miR396 and lower GRF levels. Modified GRF2, which was mutated to interfere with the interaction with miR396, was shown to be independent of miR396 regulation to which the wild-type GRF2 was subject. These plants were reported to have slightly bigger leaves than those of wild-type, however these leaves were curved downwards which could be detrimental for light capture and photosynthesis. Those results indicated that miR396 levels can significantly restrict cell proliferation in plants.
In the present disclosure, it is shown that a mutant GRF3 (sometimes referred to herein as rGRF3) and mutant GRF3 orthologues (sometimes referred to herein as rGRF3 orthologues) are relieved of miR396 regulation, and that plants comprising the mutant GRF3 or mutant GRF3 orthologues have improved productivity and/or yield (including greater leaf area, greater cell numbers, increased biomass, increased stress resistance, delayed leaf senescence, increased seed production, increased seed yield, increased root growth, increased root elongation speed and greater tolerance to drought), whether compared to wild-type plants or to plants comprising a mutant GRF2 relieved of miR396 regulation. Furthermore, the leaves from mutant GRF3 plants or mutant GRF3 orthologue plants were not curved downwards as those of mutant GRF2. The slight increase in leaf area observed in mutant GRF2 plants were caused by increasing its level at least twenty-fold compared with the level of GRF2 in wild-type plants; however, just three to five times more mutant GRF3 compared with the level of GRF3 in wild-type plants has been observed to cause a much larger impact on leaf size and plant biomass.
When the GRF3 modification or GRF3 orthologue modification is combined in a plant overexpressing GIF1, these effects are greatly enhanced.